CN1911450A - Live target adjuvant containing D-galactose and sterol or aliphatic alcohol and its preparation - Google Patents
Live target adjuvant containing D-galactose and sterol or aliphatic alcohol and its preparation Download PDFInfo
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Abstract
A synthesis of the liver-target formate derivative containing galactose structure and its preparation containing said derivative are disclosed. Said derivative is prepared through the esterifying reaction sterol or emtrol and the compound containing lactose structure by di-primary amine compound in organic solvent other than water. It can modify the liposome, emulsion, milimicron vesicle (particle) and micelles to improve the liver-targeting performance.
Description
Technical field:
The present invention relates to medical technical field, exactly relate to liver targeted auxiliary material and preparation thereof that a class contains D-galactose and sterol or aliphatic alcohol.
Background technology:
Hepatocarcinoma (HCC) is prevailing pernicious constitutional liver neoplasm, and HCC is in most of population of US and European, and is not general.But in many Asia and African country, be one of three big fatal cancers.China is the district occurred frequently of viral hepatitis, and the average year sickness rate is 120-140/10 ten thousand, and 280,000 people that have an appointment every year die from and the hepatitis diseases associated.China's hepatitis B virus carriers reaches 1.3 hundred million, and hepatitis B virus (HBV) infection rate is up to 57.63%, and promptly the whole nation has at least 600,000,000 people to infect HBV.HBsAg positive rate 9.75% has 1.2 hundred million people approximately, accounts for 1/3 of the whole world; Wherein about 1/4 will develop into chronic hepatopathy, and part patient can develop into liver cirrhosis, even develop into hepatocarcinoma.Hepatitis B is serious harm China people's health not only, but also bring serious social economic burden for country.The treatment time of chronic viral hepatitis B is long, and medical expense is higher, adds inappropriate Drug abuse again, has more increased the weight of extra financial burden.The direct medical treatment that China is used for the treatment of chronic hepatitis, liver cirrhosis, hepatocarcinoma every year expends about 30,000,000,000 yuans.Therefore, strengthen the prevention of viral hepatitis; Seeking effective Therapeutic Method of chronic hepatitis, is the current key subjects that need to be resolved hurrily.
Liver participates in many processes such as digestion in the body, drainage, detoxifcation and immunomodulating, hepatocyte mainly be divided into hepatic parenchymal cells (parenchymal cell, P) and liver non-parenchymal cell (non-parenchymalcell, NP).Hepatic parenchymal cells is a topmost cell in the hepatocyte, also is simultaneously the main cell that is subjected to invasions such as virus, mainly occurs in hepatic parenchymal cells as hepatitis and hepatocarcinoma.Asialoglycoprotein receptor (Asialoglycoprotei recepters, ASGPR) be peculiar a kind of efficient endocytosis receptor on the hepatic parenchymal cells film, can discern glycoprotein and the combination with it that molecular end has galactose residue by specificity, complex generation microscopic clusters collection, lysosome is swallowed in caving in back, disengages medicine.Asialoglycoprotein receptor is not degraded, and is transported to again on the cell membrane, participates in the next round circulation.Can utilize this glycoprotein (Gal-HSA) of doing in order to galactose residue is carrier, the development hepatic targeting drug.
Still there is not at present a kind of medicine that can rapidly, directly remove hepatitis B virus.The curative effect of at present best antiviral drugs also only can reach about 50%.Though the interventional therapy curative effect is good, need under the guiding of X line, CT and ultrasonic device the technology of utilizing specific apparatus to treat.And by can with the asialoglycoprotein receptor specific bond contain galactosyl liver targeted auxiliary material drug administration carrier, then may remove virus whole in the liver and improve gene transfering efficiency greatly, and reduce poisonous side effect of medicine, avoid the operation misery.
Lactobionic acid contains D-galactose end group, also contains the reactive group-COOH that responds, and easy and other chemical compound reaction generates liver targeted auxiliary material.It is starting material with galactose or lactose that many scholars are arranged both at home and abroad, with galactose residue and medicine (comprising albumen or polypeptide class) or carrier material coupling mutually, and preparation liver targeting vector.Takenaga etc. react in 0.1% sodium lauryl sulfate buffer with lactose lactone and alpha-interferon, make product and be applied to human body amniotic membrane/sindbis virus system, make drug level keep 55.1%-82.4%, and in mouse experiment, demonstrate very strong liver targeting polarization.(Takenaga?M，Sakurai?K，Igarashi?R，et?al.Can?PatAppl，CA，130，230；CA，123：144661)。Yang Jinxiang etc. have synthesized the galactose derivative Gal β that contains the oxygen ethyl
1-(CH2-CH2-O) 3-C14H29 modifies the cantharidin liposome with it.Show that by pharmacological evaluation cantharidin liposome that galactose modifies obviously is better than the cantharidin conventional liposome to the target function of active liver, both hold the allowance maximum in liver can differ 2.6 times, and the holdup time significant prolongation.And ethyoxyl can strengthen the binding ability of the liposome of liver galactosylated acceptor and the modification of some galactose derivatives.(Hisachi?Yoshioka.J?Pham?Sci，1993，82：273)。Mitsuru Hashida etc. synthesize cholestene-5-oxygen base-N-[4-[[1-imido grpup-2-β-D-sulfo-galactose ethyl with IME-galactoside and lentochol reaction] amino] butyl] Methanamide (Gal-C4-Chol); And with this material preparation liver target liposomes, found that the liver target liposomes that contains Gal-C4-Chol distributes than being 15.1 in hepatic parenchymal cells and nonparenchymal cell, and conventional liposome distributes in hepatic parenchymal cells and nonparenchymal cell than being 1.1.(Kawakami?S，Munakata?C，Fumoto?S，et?al.Novel?galactosylated?liposomes?for?hepatocyte-selectivetargeting?of?lipophilic?drugs.J?Pharm?Sci，2001，90(2)：105-113.)。Wang Shao rather waits and uses lactobionic acid, succinic anhydrides and cholesterol are raw material, synthesized (5-cholestene-3 β-oxygen base) 4-oxo-4-[2-lactose amide base ethylamino] butyrate, and this novel liver targeted auxiliary material prepared liver targeting peptide-doxorubicin liposome, the amycin liver targeting efficient (Te) of the glycosyl galactose liposome of discovery 10% is 3.25 times of conventional liposome, and in hepatic parenchymal cells and nonparenchymal cell, distribute than 6.77 times of (Shaoning Wang that are conventional liposome distribution ratio, Yingkun Qiu, Hui Xu, et al.Chi Chem Letter, 2006,17 (2): 173～176.).Fujita T modifies back (Gal-SOD) with superoxide dismutase (SOD) with galactose, but targeting is in hepatic parenchymal cells, and demonstrate ability (the Nishikawa M of the anti-law during ischemia damage that is better than common SOD, Tamakic, et al.J Pharmcol Exp Ther, 1992,263 (3): 971-978.).
Make reaction raw materials with galactose or lactose, need to activate earlier hydroxyl with the raising extent of reaction, and need protect non-reactive hydroxyl, so the reaction more complicated, productive rate is low.Lactobionic acid contains the galactose end group, contains the reactive group-carboxyl that responds simultaneously, easy and other chemical compound generation acylation reaction.
Summary of the invention:
The purpose of this invention is to provide the liver targeted auxiliary material of a kind of D-of containing galactose and sterol or aliphatic alcohol, and be applied in the drug administration carrier, to realize the needs of liver targeting.The objective of the invention is to realize that by following scheme it is that raw material is made with the chloromethyl ester of lactobionic acid, diamine and sterol or aliphatic alcohol, it is characterized in that general structure is as follows:
In the formula:
R
1Be sterol or aliphatic alcohols chemical compounds such as cholesterol, sitosterol.
R
2Straight or branched alkyl, substituted alkyl, aromatic radical or substituted aromatic base for 2-10 carbon atom; Polyethylene Glycol-CH
2(CH
2OCH
2)
nCH
2-, n=1-200); Amino-acid alkyl ester (aryl ester).
Taked following measure in order to obtain target compound:
1, synthetic (2-aminoethyl base) formic acid cholesteryl ester (is example with ethylenediamine and cholesterol chloromethyl ester),
Obtain target compound with the lactobionic acid lactone reaction again
The cholesterol chloromethyl ester dissolves with dichloromethane, puts in the charging hopper, is added drop-wise to excessive 1-10 ethylenediamine CH doubly
2Cl
2In the solution, stirring reaction 10hr under the ice-water bath.Remove CH under reduced pressure
2Cl
2, add water 20mL (ethylenediamine and hydrochlorate thereof that the flush away unreacted is intact), mixing, reuse CH
2Cl
2(15ml * 2) extraction, deionization washing (15ml * 2), an amount of anhydrous Na of reuse
2SO
4Dried overnight.The pressure reducing and steaming solvent gets cured shape white solid, i.e. (2-aminoethyl base) formic acid cholesteryl ester.
Get lactobionic acid and add dissolve with methanol, 50 ℃ of reduction vaporizations are removed methanol, adding 2～10 times of volume of ethanol, behind the ultra-sonic dispersion again reduction vaporization remove ethanol, repeat 3～10 times after, place 70～80 ℃ of baking ovens to place 12～48hr its solid, promptly get the lactobionic acid lactone.
Dissolve in the lactobionic acid lactone adding polar solvent, (2-aminoethyl base) formic acid cholesteryl ester dissolves 0～100 ℃ of stirring reaction 1～36h with non-polar solven.Negate answers mixture to remove partial solvent in 30～80 ℃ of reduction vaporizations, removes by filter precipitation, and 2～10 times of filtrate adding distil water dilutions place diluent the 12～72h that dialyses in the bag filter again, concentrate dialysate, and lyophilizing gets product.
2, synthetic (2-aminoethyl base) formic acid cholesteryl ester obtains target compound with the reaction of N-(lactose acyloxy) imidodicarbonic diamide again
(2-aminoethyl base) formic acid cholesteryl ester synthetic the same.
Synthetic lactobionic acid active ester.Lactobionic acid is dissolved among the DMF, be cooled to-5 ℃～-30 ℃, add N, N ＇-dicyclohexylcarbodiimide, in-10 ℃～-20 ℃ stirring reaction 10～120min, rise to-5 ℃ of reaction 5～60min, add N-hydroxy-succinamide then, in-5 ℃ of stirring reaction 0.5～2h, continue to stir 6～36h in room temperature.With reactant mixture elimination 1,3-Dicyclohexylurea, gained filtrate evaporate to dryness adds dehydrated alcohol, drips ether behind the ultra-sonic dispersion, separates out white floccule, again through sucking filtration, and the ether washing, vacuum drying gets white amorphous products.
Get in the lactobionic acid active ester adding polar solvent and dissolve, (2-aminoethyl base) formic acid cholesteryl ester dissolves 0～100 ℃ of stirring reaction 1～36h with non-polar solven.Negate answers mixture to remove partial solvent in 30～80 ℃ of reduction vaporizations, removes by filter precipitation, and 2～10 times of filtrate adding distil water dilutions place diluent the 12～72h that dialyses in the bag filter again, concentrate dialysate, and 50～90 ℃ of evaporated under reduced pressure get product.
Wherein polar solvent comprises: methanol, dimethyl sulfoxide, dimethyl formamide, N-methylformamide, dimethyl acetylamide, N-Methyl pyrrolidone etc.Non-polar solven comprises: dichloromethane, chloroform, ethyl acetate, petroleum ether, oxolane, acetone, toluene, dioxane, ether etc.
3, prepare the aliphatic alcohol chloromethyl ester earlier, resynthesis (2-aminoethyl base) formic acid docosane alcohol ester (is example with ethylenediamine and tadenan) obtains target compound with the lactobionic acid lactone reaction again
Tadenan adds the trichloroethylene dissolving, other gets triphosgene and is dissolved in 2～50 times of trichloroethylenes and 0.1～5 times of DMF, and 0.5～5h dropwises under refluxing, and continues reaction 2～12h, remove by filter insoluble matter, remove excessive solvent under reduced pressure, residue steams and removes the part cyclohexane extraction with cyclohexane extraction extract return method 5～60min, filtered while hot is removed insoluble matter, frozen water cooling filtrate is separated out the product crude product, gets tadenan chloromethyl ester white solid through recrystallization.
The tadenan chloromethyl ester dissolves with dichloromethane, puts in the charging hopper, is added drop-wise to excessive 1-10 ethylenediamine CH doubly
2Cl
2In the solution, stirring reaction 10hr under the ice-water bath.Remove CH under reduced pressure
2Cl
2, add water 20mL (ethylenediamine and hydrochlorate thereof that the flush away unreacted is intact), mixing, reuse CH
2Cl
2(15ml * 2) extraction, deionization washing (15ml * 2), an amount of anhydrous Na of reuse
2SO
4Dried overnight.The pressure reducing and steaming solvent gets white solid, i.e. (2-aminoethyl base) formic acid docosane alcohol ester.
The preparation of lactobionic acid lactone is the same.Dissolve in the lactobionic acid lactone adding polar solvent, (2-aminoethyl base) formic acid docosane alcohol ester adds non-polar solven dissolving, 0～100 ℃ of stirring reaction 1～36h.Negate answers mixture to remove partial solvent in 30～80 ℃ of reduction vaporizations, removes by filter precipitation, and 2～10 times of filtrate adding distil water dilutions place diluent the 12～72h that dialyses in the bag filter again, concentrate dialysate, and lyophilizing gets product.
Wherein polar solvent comprises: methanol, ethanol, dimethyl sulfoxide, dimethyl formamide, trichloroethylene, N-methylformamide, dimethyl acetylamide, N-Methyl pyrrolidone etc.Non-polar solven comprises: dichloromethane, chloroform, ethyl acetate, petroleum ether, oxolane, acetone, toluene, dioxane, ether etc.
Utilize the characteristics of the existing targeting group-galactosyl of lactobionic acid and reaction active groups-carboxyl; obtain a kind of liver target direction amphipathic nature adjuvant with referring to fat alcohol or sterol through serial esterification and acylation reaction, the lipid end group can be used for the targeting of drug administration carriers such as liposome, Emulsion, nanoparticle, microsphere, nanocapsule, micelle.This reaction mechanism mechanism of reaction is short, and avoided some poisonous reagent as the application of red phosphorus, bromine, sodium hydride etc.; The adjuvant that reaction obtains contains ester bond and amido link, easily degraded in the body.
This liver targeted auxiliary material can be used to drug administration carriers such as modified liposome, Emulsion, nanoparticle, microsphere, nanocapsule and micelle, and content can reach the 0.1-100% (mol%) of lipid components, is used for the targeted therapy of anti-hepatocarcinoma, hepatitis medicament.
Description of drawings:
The synthetic route of Fig. 1 [(2-lactose amide base) ethylamino-] formic acid cholesteryl ester (CH-ED-LA)
Fig. 2 amycin aqueous solution (Dox solution), conventional liposome (Dox-CL) and target liposomes (Gal-DOX-L) distribute in the mice plasma regulating liver-QI
Fig. 3 zidovudine aqueous solution (AZT solution), the common nano-emulsion of azidothymidine palmitate (AZTP-NE) and azidothymidine palmitate targeted nano Emulsion (Gal-AZTP-NE) distribute in the mice plasma regulating liver-QI
Fig. 4 zidovudine aqueous solution (AZT solution), azidothymidine palmitate conventional liposome (AZTP-CL) and azidothymidine palmitate target liposomes (Gal-AZTPL) distribute in the mice plasma regulating liver-QI
The specific embodiment:
Below in conjunction with example the present invention is done further detailed description
Embodiment 1:[(2-lactose amide base) ethylamino-] formic acid cholesteryl ester (CH-ED-LA) synthetic
By synthetic (2-aminoethyl base) formic acid cholesteryl ester, obtain target compound [(2-lactose amide base) ethylamino-] formic acid cholesteryl ester (CH-ED-LA) with the lactobionic acid lactone reaction again.See accompanying drawing 1.
1. the lactobionic acid lactone is synthetic
Get lactobionic acid and add dissolve with methanol, 50 ℃ of reduction vaporizations are removed methanol, repeat 3～4 times after, place 70～80 ℃ of baking ovens to place 48hr its solid, promptly get the lactobionic acid lactone.
2. (2-aminoethyl base) formic acid cholesteryl ester (I)
Get 0.45g cholesterol chloromethyl ester (1mmol), use 60mL CH
2Cl
2Dissolving is put in the Dropping funnel, slowly joins to contain 5.84g ethylenediamine (100mmol) CH
2Cl
2In the solution, stirring reaction 10hr under the ice-water bath.Remove CH under reduced pressure
2Cl
2, add water 20mL (ethylenediamine and hydrochlorate thereof that the flush away unreacted is intact), mixing, reuse CH
2Cl
2(15ml * 2) extraction, deionization washing (15ml * 2), an amount of anhydrous Na of reuse
2SO
4Dried overnight.The pressure reducing and steaming solvent gets cured shape white solid (I) 0.48g, (productive rate is 91.1%), TLC (methylene chloride=9: 1): R
f=0.2.FTIR (cm
-1): 3339.2,1551.2 (NH), 2939.3 (cyclenes CH), 1716.7,1696.8,1276.9,1136.8 (NHCOOR) ESI-MS (m/z): 474.1 (M+H)
+, 495.3 (M+Na)
+
3. (2-lactose amide base) ethylamino-] formic acid cholesteryl ester (II)
Get lactobionic acid lactone 0.52g (1.53mmol), add dimethyl sulfoxide 8mL, stirring and dissolving under 40 ℃ of water-baths, dropwise add the dissolved Compound I solution of dichloromethane (0.48g, 1mmol), drip 3～5 triethylamines again, be warming up to 50 ℃ of back flow reaction 36hr then.Dichloromethane is removed in decompression, and sucking filtration is considered, disgorging, and filtrate adds the distilled water of 2 times of volumes, places the bag filter 48hr that dialyses, and removes dimethyl sulfoxide and unreacted lactobionic acid, with the dialysis solution evaporate to dryness.Add dichloromethane/dehydrated alcohol mixed solvent 20mL again, the ultrasonic dissolution solid filters and removes insoluble matter, behind the filtrate evaporate to dryness, gets end-product CH-ED-LA (0.44g, productive rate 53.7%).TLC (ethyl acetate/ethanol/water=6: 4: 1, aniline/diphenylamines/85% phosphoric acid developer), Rf=0.4.FTIR (cm
-1): 3327.3,1626.1,1576.8 (NH), 2939.3 (cyclenes CH), 1696,1245,891.4 (NHCOOR).HPLC-DAD: area normalization method is calculated, and degree of purity of production is greater than 90%.ESI-MS(m/z)：835.5(M+Na)
+，
1H?NMR(C
5D
5N，ppm)：8.13(brt，1H，J＝6.8，-NH)，7.99(brt，2H，J＝6.8，-NH)，5.37(t?1H，H-6)，5.20(d，1H，J＝6.7，H-1″)，5.18(d，1H，J＝4.26，H-2＇)，4.84(m，1H，H-3)，4.78(m，1H，H-4′)，4.70(m，1H，H-5′)，
13C?NMR(C
5D
5N，δppm)：174.79(C-1′)，157.35(C-1)，140.45(C-5)，122.57(C-6)，106.54(C-1″)。
Embodiment 2:(2-lactose amide base) ethylamino-] formic acid cholesteryl ester (CH-ED-LA) synthetic
By synthetic N-(lactose acyloxy) imidodicarbonic diamide, obtain [(2-lactose amide base) ethylamino-] formic acid cholesteryl ester (CH-ED-LA) with the reaction of (2-aminoethyl base) formic acid cholesteryl ester again.
1. synthetic lactobionic acid active ester
(5mmol) is dissolved among the 30mLDMF with the 1.80g lactobionic acid, be cooled to-15 ℃～-20 ℃, add 3.10gN, N ＇-dicyclohexylcarbodiimide (15mmol), behind-15 ℃ of stirring reaction 20min, rise to-5 ℃ of reaction 15min, add 1.15gN-N-Hydroxysuccinimide (10mmol) then, in-5 ℃ of stirring reaction 1h, at room temperature stir 25h.With reactant mixture elimination 1,3-Dicyclohexylurea, gained filtrate evaporate to dryness adds the 20mL dehydrated alcohol to about 5mL, drips ether behind the ultra-sonic dispersion, separate out white floccule, through sucking filtration, the washing of 2mL ether is behind the vacuum drying again, get white powder 2.05g (productive rate 90%), TLC (dichloromethane/ethanol=1: 1, aniline/diphenylamines/85% phosphoric acid developer), R
f=0.61.FTIR(cm
-1)：3326.6，2929.3(br，OH)，1627.5(imide，C＝O)，1574.7，1271.4(RCOON)，1088.2(C-O-C)。
2. synthetic [(2-lactose amide base) ethylamino-] formic acid cholesteryl ester (II)
Get lactobionic acid active ester 0.46g (1mmol), add dimethyl sulfoxide 3mL, after 40 ℃ of stirring in water bath dissolvings, (0.28g, 0.6mmol) 10mL drip 2～4 triethylamines again, are warming up to 50 ℃ of back flow reaction 12hr dropwise to add the dichloromethane solution of Compound I.Dichloromethane is removed in decompression then, and sucking filtration gets light yellow DMSO solution, adds the distilled water of 2 times of volumes in filtrate, and ultrasonic 1min is placed on dialysis 48hr in the bag filter, removes DMSO and remaining lactose activity ester.With the dialysis solution evaporate to dryness, add dichloromethane/dehydrated alcohol mixed solvent 20mL more at last, the ultrasonic dissolution solid, sucking filtration is removed insoluble matter, behind the filtrate evaporate to dryness, gets end-product II (0.41g, productive rate 50%).
Embodiment 3:[(2-lactose amide base) hexylamine base] formic acid cholesteryl ester (CH-HD-LA) synthetic
By synthetic (the amino hexylamine base of 2-) formic acid cholesteryl ester, obtain [(2-lactose amide base) hexylamine base] formic acid cholesteryl ester (CH-HD-LA) again with the lactobionic acid lactone reaction.
Get 0.45g cholesterol chloromethyl ester (1mmol), use 60mL CH
2Cl
2Dissolving is put in the Dropping funnel, slowly joins to contain 11.6g hexamethylene diamine (100mmol) CH
2Cl
2In the solution, stirring reaction 10hr under the ice-water bath.Remove CH under reduced pressure
2Cl
2, add water 20mL (hexamethylene diamine and hydrochlorate thereof that the flush away unreacted is intact), mixing, reuse CH
2Cl
2(15ml * 2) extraction, deionization washing (15ml * 2), an amount of anhydrous Na of reuse
2SO
4Dried overnight.The pressure reducing and steaming solvent gets gray solid 0.43g, (productive rate is 82.1%), TLC (methylene chloride=9: 1); R
f=0.3.FTIR (cm
-1): 3341.2,1561.4 (NH), 2938.6 (cyclenes CH), 1715.7,1676.4 (C=O), 1277.1 (C-N).ESI-MS(m/z)：528.5(M+H)
+，551.7(M+Na)
+。
Get lactobionic acid lactone 0.52g (1.53mmol), add dimethyl sulfoxide 8mL, stirring and dissolving under 40 ℃ of water-baths, dropwise add dichloromethane dissolved (the amino hexylamine base of 2-) formic acid cholesteryl ester solution (0.53g, 1mmol), drip 3～5 triethylamines again, be warming up to 50 ℃ of back flow reaction 36hr then.Dichloromethane is removed in decompression, sucking filtration, and disgorging, filtrate adds the distilled water of 2 times of volumes, places the bag filter 48hr that dialyses, and removes dimethyl sulfoxide and unreacted lactobionic acid, with the dialysis solution evaporate to dryness.Add dichloromethane/dehydrated alcohol mixed solvent 20mL again, the ultrasonic dissolution solid, sucking filtration is removed insoluble matter, behind the filtrate evaporate to dryness, gets end-product CH-HD-LA (0.44g, productive rate 53.7%).TLC (ethyl acetate/ethanol/water=6: 4: 1, aniline/diphenylamines/85% phosphoric acid developer), R
f=0.46.FTIR (cm
-1): 3327.7,627.5,1561.2 (NH), 12940.6 (cyclenes CH), 1694.5 (NHCOOR).ESI-MS(m/z)：868.6(M+Na)
+。
13C?NMR(C
5D
5N，δppm)：174.7(COON)，157.6(NCOOR)。
Embodiment 4:[(2-lactose amide base) ethylamino-] formic acid docosane alcohol ester (DS-ED-LA) synthetic
1. synthetic tadenan chloromethyl ester
Tadenan adds the trichloroethylene dissolving, other gets triphosgene and is dissolved among 2～50 times of trichloroethylenes and 0.1～5 times of DMF, and 0.5～5h dropwises under refluxing, and continues reaction 2～12h, remove by filter insoluble matter, remove excessive solvent under reduced pressure, residue steams and removes the part cyclohexane extraction with cyclohexane extraction extract return method 5～60min, filtered while hot is removed insoluble matter, frozen water cooling filtrate is separated out the product crude product, gets tadenan chloromethyl ester white solid through recrystallization.
2. synthetic (2-aminoethyl base) formic acid docosane alcohol ester
The tadenan chloromethyl ester dissolves with dichloromethane, puts in the charging hopper, is added drop-wise in excessive 1～10 times ethylenediamine dichloromethane solution stirring reaction 10hr under the ice-water bath.Remove CH under reduced pressure
2Cl
2, add water 20mL (ethylenediamine and hydrochlorate thereof that the flush away unreacted is intact), mixing, reuse dichloromethane (15ml * 2) extraction, deionization washing (15ml * 2), an amount of anhydrous Na of reuse
2SO
4Dried overnight.The pressure reducing and steaming solvent gets white solid, i.e. (2-aminoethyl base) formic acid docosane alcohol ester.TLC (methylene chloride=2: 1): R
f=0.45.FTIR(cm
-1)：2958.4，2931.2，2878.3(br，CH)，1776.7(C＝O)，1728.1(COOR)。ESI-MS(m/z)：388.6(M+H)
+。
3. synthetic [(2-lactose amide base) ethylamino-] formic acid docosane alcohol ester (DS-ED-LA)
The preparation of lactobionic acid lactone is the same.Dissolve in the lactobionic acid lactone adding polar solvent, (2-aminoethyl base) formic acid docosane alcohol ester adds non-polar solven dissolving, 0～100 ℃ of stirring reaction 1～36h.Negate answers mixture to remove partial solvent in 30～80 ℃ of reduction vaporizations, removes by filter precipitation, and 2～10 times of filtrate adding distil water dilutions place diluent the 12～72h that dialyses in the bag filter again, concentrate dialysate, and lyophilizing gets product.TLC (methylene chloride=2: 1): R
f=0.4.FTIR(cm
-1)：3274.7，2875.3(br，NH，CH)，15671.2(NH)，1687.2(NHCOOR)。ESI-MS(m/z)：776.1(M+Na)
+。
13C?NMR(C
5D
5N，δppm)：175.4(COON)，169.5(NCOOR)。
Embodiment 5: distribute in the preparation of amycin liver target liposomes and the body
Amycin (doxorubicin) is one one a line medicine of treatment hepatocarcinoma, is clinical antitumor drug commonly used, and antitumor spectra is wide, good effect.Yet this medicine also can produce the serious adverse reaction of general in killing tumor cell, after one's own heart dysentery, bone marrow depression etc., and therapeutic index is low, and clinical practice is restricted.Adopt the liver target liposomes of material preparation of the present invention, can improve the concentration of medicine in liver, to improve anti-liver cancer efficacy.
The preparation of liposome:
Prescription is formed:
Amycin 0.10g
HSPC 5.0g
Cholesterol (CH) 1.7g
CH-ED-LA 0.30g
Sulphuric acid amine 3.3g
Water adds to 100mL
HSPC, CH, the CH-ED-LA of recipe quantity are added in the dispensing containers, use an amount of dissolve with ethanol under 60 ℃ of conditions, and wave most ethanol film forming, the synthermal ammonium sulfate buffer aquation that adds 300mM down, preparation blank liposome.Blank liposome is handled through the microjet instrument, by 0.22 μ m microporous filter membrane granulate.Liposome behind the granulate is inserted in the bag filter, uses normal saline dialysis, removes the ammonium sulfate of outer water, obtains the gradient liposome.Get amycin solution and mix by a certain percentage,, promptly obtain amycin liver target liposomes (1mg/mL) in 60 ℃ of hatching 30min with the gradient liposome.Granularity is: 50～350nm.Particle mean size 89nm.
Distribute in the body:
Get Kunming mouse, body weight 18～22g, divide 3 groups: amycin aqueous solution, conventional liposome group and target liposomes group, press the dosage tail intravenously administrable of 10mg/kg, get blood in different time points through the eye socket vein, the sacrificed by decapitation animal, the result shows that the concentration of target liposomes group amycin in blood plasma significantly reduces, and the concentration in liver significantly improves (seeing accompanying drawing 2), the AUC in liver
0-4Be 3 times of conventional liposome.
Distribute in the preparation of embodiment 6 azidothymidine palmitate liver-targeted nanometer Emulsions and the body
Behind the human infection HIV, liver is the organ that very easily suffers damage, and is infecting the later stage, and liver more becomes one of deep layer storage storehouse of HIV, and conventional formulation is difficult to remove the HIV virus that residues in the liver.(Zidovudine, AZT) as one of anti-HIV drug of first choice, its determined curative effect can reduce opportunistic infection to zidovudine, delays the development of AIDS related complex (ARC) to AIDS.AZTP is the cetylate derivant of AZT, utilizes this Palmic acid fat chain to insert in the nano-emulsion.On the basis of realizing the passive target administration, in original prescription, add liver targeted auxiliary material of the present invention, preparation has the initiatively glycosyl galactose nano-emulsion of liver targeting, and medicine is concentrated in liver, helps removing the HIV virus that residues in the liver.
The preparation of nano-emulsion
Prescription is formed:
AZTP 0.30g
Midchain oil 5g
Lecithin 2g
TPGS 0.50g
Glycerol 2g
CH-ED-LA 0.30g
Water adds to 100mL
The midchain oil, lecithin and the TPGS that take by weighing recipe quantity place small beaker, and 70 ℃ of water-bath high speed dispersion machine 14000l/min stir down and make its dispersing and dissolving, and the AZTP that adds recipe quantity then continues to stir makes its dissolving; In addition the glycerol of recipe quantity and 6.0mL water are placed under the synthermal water-bath magnetic agitation and dissolve, as water; Under dispersion machine 14000l/min stirred, gradation slowly joined water in the oil phase, disperses 15min then under 10000l/min, be cooled to room temperature after, add water and be settled to 30mL, promptly get colostrum.With colostrum with the microjet instrument with 70psi pressure homogenizing 8 times, fill, inflated with nitrogen is jumped a queue, and adds the aluminium lid sealing, flowing steam sterilization 30min, promptly.
Distribute in pharmacokinetics and the body
Get 72 of Kunming kind white mice, be divided into 2 groups at random, press 30mgkg
-1Dosage is tail vein injection AZT solution, the common nano-emulsion of AZTP and Gal-AZTP nano-emulsion respectively, in 1,5,10,15,20,30,40 and 60min (every 3 mices) get blood through the eye socket vein, dislocation is put to death the back and is taken out liver, clean with normal saline, filter paper blots, and weighs, get 0.1g through homogenate, behind the protein precipitation, carry out HPLC and analyze, measure the concentration of zidovudine.Pharmaceutical concentration-time curve in the blood regulating liver-QI is seen accompanying drawing 3.
Distribute in the preparation of embodiment 7 azidothymidine palmitate liver target liposomes and the body
Liposome is a kind of very promising drug carrier system, and its bilayer similar cell membrane has good cell affinity and histocompatibility.Cholesterol is the basic substance that constitutes liposome membrane, novel liver targeted auxiliary material of the present invention contains cholesterol or lipid end group, can be easy to insert in the liposome phospholipid bilayer film, D-galactose end group stretches out at surface of liposome, pastille liposome guiding liver parenchyma with preparation, medicine is concentrated in liver, help removing the HIV virus that residues in the liver, improve therapeutic effect.
The preparation of AZTP liposome
Prescription is formed:
AZTP/mg 60
Epikuron?170/mg 1000
Cholesterol/mg 125
Liver targeted auxiliary material/mg is an amount of
5mmolL
-1PBS (pH7.0) adds to 20mL
Alcohol injection is dissolved in phospholipid, cholesterol, liver targeted auxiliary material and the AZTP (medicine fat ratio is 1: 16.7) of recipe quantity in an amount of dehydrated alcohol, slowly drips alcoholic solution in the PBS of 50 ℃ of constant temperature magnetic agitation (5m molL with syringe
-1, pH7.0 presses 2000 editions two appendix XV D preparations of Chinese Pharmacopoeia) in, stir 5min, afterwards temperature is adjusted downward to 40 ℃ and continues to stir, hatching 10min.With the liposome that makes with above-mentioned PBS standardize solution.After being cooled to room temperature, ultra-sonic dispersion under the probe of cell breakage instrument, power are 600w * 8min (work 10s, intermittently 10s).After 0.8,0.45 and 0.22 μ m microporous filter membrane granulate successively, the AZTP liposome, standby.
Distribute in pharmacokinetics and the body
Get 72 of Kunming kind white mice, be divided into 2 groups at random, press 30mgkg
-1Dosage is tail vein injection AZT solution, AZTP conventional liposome and Gal-AZTP liposome respectively, in 1,3,5,7,10,15,20 and 30min (every 3 mices) get blood through the eye socket vein, dislocation is put to death the back and is taken out liver, clean with normal saline, filter paper blots, and weighs, get 0.1g through homogenate, behind the protein precipitation, carry out HPLC and analyze, measure the concentration of zidovudine.Pharmaceutical concentration-time curve in the blood regulating liver-QI is seen accompanying drawing 4.
Claims (9)
1, a kind of liver targeted auxiliary material that contains D-galactose and sterol or aliphatic alcohol, it is characterized in that: its general structure of this liver targeted auxiliary material is as follows:
In the formula:
R
1Be sterol or aliphatic alcohols chemical compounds such as cholesterol, sitosterol
R
2Straight or branched alkyl, substituted alkyl, aromatic radical or substituted aromatic base for 2-10 carbon atom; Polyethylene Glycol-CH
2(CH
2OCH
2)
nCH
2-, n=1-200; Amino-acid alkyl ester.
2, a kind of liver targeted auxiliary material that contains D-galactose structure according to claim 1 is characterized in that: it is that diamine by the sugar that contains D-galactose end group, sterol or aliphatic alcohol and a series of two ends amino-contained makes through acylation reaction.
3, a kind of liver targeted auxiliary material that contains D-galactose structure according to claim 2 is characterized in that: contained D-galactose structure is selected from the sugar that contains D-galactose end group, lactobionic acid, lactobionic acid lactone and lactobionic acid active ester; Sterol comprises cholesterol and sitosterol or sterin; Aliphatic alcohol refers to that the C number is 6～50 saturated or unsaturated fatty alcohol; R in the diamine
2Can be C
2～C
20Straight or branched alkyl, substituted alkyl, aromatic radical and substituted aromatic base, or be oxygen ethyl (CH
2-CH
2-O-CH
2-CH
2)
n, be polyethylene glycol diamines, wherein n=1～100; Aminoalkyl ester.
4, a kind of a kind of preparation method that contains the liver targeted auxiliary material of D-galactose structure and sterol as claimed in claim 1 is characterized in that:
The cholesterol chloromethyl ester dissolves with dichloromethane, puts in the charging hopper, is added drop-wise to excessive 1-10 diamine CH doubly
2Cl
2In the solution, stirring reaction 10hr under the ice-water bath.Remove CH under reduced pressure
2Cl
2, add water 20mL (ethylenediamine and hydrochlorate thereof that the flush away unreacted is intact), mixing, reuse CH
2Cl
2(15ml * 2) extraction, deionization washing (15ml * 2), an amount of anhydrous Na of reuse
2SO
4Dried overnight.The pressure reducing and steaming solvent gets cured shape white solid, i.e. diamine list formic acid cholesteryl ester;
Get lactobionic acid and add dissolve with methanol, 50 ℃ of reduction vaporizations are removed methanol, adding 2～10 times of volume of ethanol, behind the ultra-sonic dispersion again reduction vaporization remove ethanol, repeat 3～10 times after, place 70～80 ℃ of baking ovens to place 12～48hr its solid, promptly get the lactobionic acid lactone;
Dissolve in the lactobionic acid lactone adding polar solvent, diamine list formic acid cholesteryl ester adds non-polar solven dissolving, 0～100 ℃ of stirring reaction 1～36h.Negate answers mixture to remove partial solvent in 30～80 ℃ of reduction vaporizations, removes by filter precipitation, and 2～10 times of filtrate adding distil water dilutions place diluent the 12～72h that dialyses in the bag filter again, concentrate dialysate, and lyophilizing gets product.
5, a kind of a kind of preparation method that contains the liver targeted auxiliary material of D-galactose structure and aliphatic alcohol as claimed in claim 1, it is characterized in that: aliphatic alcohol adds the trichloroethylene dissolving, other gets triphosgene and is dissolved in 2～50 times of trichloroethylenes and 0.1～5 times of DMF, 0.5～5h dropwises under refluxing, continue reaction 2～12h, remove by filter insoluble matter, remove excessive solvent under reduced pressure, residue cyclohexane extraction extract return method 5～60min, steam and remove the part cyclohexane extraction, filtered while hot is removed the insoluble noon, frozen water cooling filtrate, separate out the product crude product, get aliphatic alcohol chloromethyl ester white solid through recrystallization;
The aliphatic alcohol chloromethyl ester dissolves with dichloromethane, puts in the charging hopper, is added drop-wise to excessive 1-10 diamine CH doubly
2Cl
2In the solution, stirring reaction 10hr under the ice-water bath.Remove CH under reduced pressure
2Cl
2, add water 20mL (ethylenediamine and hydrochlorate thereof that the flush away unreacted is intact), mixing, reuse CH
2Cl
2(15ml * 2) extraction, deionization washing (15ml * 2), an amount of anhydrous Na of reuse
2SO
4Dried overnight.The pressure reducing and steaming solvent gets white solid, i.e. diamine list formic acid aliphatic alcohol ester;
The preparation of lactobionic acid lactone is the same, dissolve in the lactobionic acid lactone adding polar solvent, diamine list formic acid aliphatic alcohol ester adds the non-polar solven dissolving, 0～100 ℃ of stirring reaction 1～36h, and negate answers mixture to remove partial solvent in 30～80 ℃ of reduction vaporizations, remove by filter precipitation, 2～10 times of filtrate adding distil water dilutions place diluent the 12～72h that dialyses in the bag filter, concentrate dialysate again, lyophilizing gets product.
6, according to the preparation method of claim 4 or 5 described liver targeted auxiliary materials, it is characterized in that: polar solvent comprises: methanol, dimethyl sulfoxide, dimethyl formamide, N-methylformamide, dimethyl acetylamide, N-Methyl pyrrolidone; Non-polar solven comprises: dichloromethane, chloroform, ethyl acetate, petroleum ether, oxolane, acetone, toluene, dioxane, ether.
7, a kind of liver targeted auxiliary material preparation that contains D-galactose and sterol or aliphatic alcohol, it is characterized in that: the preparation that contains galactosamine cholesteryl ester derivant liver targeted auxiliary material, targeting ingredient as liposome, Emulsion, nanoparticle, microsphere, nanocapsule, micelle administration carrier, be used for liver targeting, the transfection of liver target gene of medicine, its liver targeted auxiliary material accounts for 0.1～100% of lipid components in the carrier.
8, the liver targeted auxiliary material preparation that contains D-galactose and sterol or aliphatic alcohol according to claim 7, it is characterized in that: described medicine comprises: antitumor drug, anti-hepatitis virus medicament, special medication and genomic medicine in anti-hepatitis ancillary drug, the liver.
9, the liver targeted auxiliary material preparation that contains D-galactose and sterol or aliphatic alcohol according to claim 8, it is characterized in that: antitumor drug comprises: zidovudine, lamivudine, amycin, NSC-118218, vinblastine, vincristine, colchicine, Radix Salviae Miltiorrhizae, paclitaxel, Docetaxel, camptothecine, hydroxy camptothecin, topotecan, ginseng polysaccharide, astragalus polysaccharides, cisplatin, carboplatin, plug are for group, ftorafur/uracil, oxaliplatin, interferon, interleukin-2, tumor necrosis factor and adenosine triphosphate; Anti-hepatitis virus medicament comprises: acyclovir, ganciclovir, ribavirin, adefovirdipivoxil, adefovir ester, glucurolactone, think of are for card Wei, alpha-interferon, cucurbitacin, matrine, kurarinone, cantharidin, norcantharidin and nor-Cantharidic acid.; Special medication comprises in anti-hepatitis ancillary drug and the liver: inosine, hepatocyte growth factor, ubiquinone
10, coenzyme A, oleanolic acid, glycyrrhizic acid, glycyrrhizic acid monoammonium, glycyrrhizic acid diamidogen, bifendate, Malotilate, silibinin, bicyclol, propagermanium, diisopropylamine ascorbate and vitamin A, E, B and K
1
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| CN102241724A (en) * | 2010-05-11 | 2011-11-16 | 上海开拓者医药发展有限公司 | Oleanolic acid salt and its preparation method and crystal |
| US20120189571A1 (en) * | 2009-02-04 | 2012-07-26 | The Brigham And Women's Hospital., Inc. | Nanoscale platinum compounds and methods of use thereof |
| CN108588006A (en) * | 2018-05-10 | 2018-09-28 | 华东理工大学 | A kind of biological support and its preparation method and application for liver cell dimensional culture |
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| CN112778389A (en) * | 2019-12-31 | 2021-05-11 | 嘉应学院医学院 | Targeting ligand molecule, preparation method thereof and drug loading system |
| CN113274506A (en) * | 2013-12-05 | 2021-08-20 | 念·吴 | Drug delivery technology of polymer-carbohydrate conjugates |
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| US20120189571A1 (en) * | 2009-02-04 | 2012-07-26 | The Brigham And Women's Hospital., Inc. | Nanoscale platinum compounds and methods of use thereof |
| US9393227B2 (en) * | 2009-02-04 | 2016-07-19 | The Brigham And Women's Hospital, Inc. | Nanoscale platinum compounds and methods of use thereof |
| US10512696B2 (en) | 2009-02-04 | 2019-12-24 | The Brigham And Women's Hospital, Inc. | Nanoscale platinum compounds and methods of use thereof |
| CN102241724A (en) * | 2010-05-11 | 2011-11-16 | 上海开拓者医药发展有限公司 | Oleanolic acid salt and its preparation method and crystal |
| CN102241724B (en) * | 2010-05-11 | 2015-12-09 | 上海开拓者医药发展有限公司 | A kind of oleanolic acid salt and preparation method thereof and crystal |
| CN113274506A (en) * | 2013-12-05 | 2021-08-20 | 念·吴 | Drug delivery technology of polymer-carbohydrate conjugates |
| CN108588006A (en) * | 2018-05-10 | 2018-09-28 | 华东理工大学 | A kind of biological support and its preparation method and application for liver cell dimensional culture |
| CN112638425A (en) * | 2018-08-27 | 2021-04-09 | 斯玛特迪利弗利有限责任公司 | PKC inhibitors for targeted treatment of septic cholestasis by CTM |
| CN112778389A (en) * | 2019-12-31 | 2021-05-11 | 嘉应学院医学院 | Targeting ligand molecule, preparation method thereof and drug loading system |
| CN115518001A (en) * | 2022-09-05 | 2022-12-27 | 吴敏 | Cosmetic containing active polypeptide and preparation method thereof |
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